Autophagy, the cellular process of self-degradation that maintains energy balance during times of stress and eliminates harmful materials, is also required for longevity in many invertebrate models of aging. Importantly, dysregulation of autophagy is also associated with age-related diseases in humans, including central nervous system (CNS) disorders such as, Alzheimer's Disease (AD). Although, activation of autophagy has shown promise in several preclinical models of AD, the existing chemical activators are non-specific, have multiple side-effects and none target the enzymes that execute the pathway. Given that autophagy activation may be disease modifying in AD, there is a pressing need for the development of new probes that target specific enzymes within the pathway. The UNC-51-like kinase-1 (Ulk1) is a central upstream regulator of the autophagy pathway - effectively an on/off switch. Under the auspices of GM113872, our research team has recently completed a high throughput Ulk1 in vitro biochemical screening campaign and identified a series of small molecule Ulk1 activators, from four chemically-tractable structural classes. Using structure based drug design and an iterative medicinal chemistry approach we will optimize and prioritize promising new leads to deliver potent, highly selective, and CNS-penetrant Ulk1 activators. These compounds will then be used as in vivo molecular probes to facilitate interrogation of the role of autophagy in AD. To achieve this, we have established a rigorous research operating plan (ROP) employing a series of essential biochemistry, cell biology, functional and mechanistic critical path assays that will facilitate effective triaging of compounds. Following optimization of cellular potency, using cell-based cytotoxicity and autophagy flux assays, we will assess the effects of lead compounds on autophagy in C. elegans, and establish specificity with Cas9/CRISPR-based affinity site mutagenesis studies in vivo. Highly selective and potent Ulk1 activators, with good rodent CNS exposure and pharmacokinetic properties will be tested for biological activity in mice. Specifically, we will measure phosphorylation of ATG13, a direct downstream substrate of Ulk1, and modulation of autophagy flux by immune-fluorescent microscopy of LC3-tandom reporters. Compounds providing increased autophagy flux with extended pharmacokinetic/pharmacodynamic (PK/PD) relationship will then be examined in mice for safety, and toxicity. Finally, the most active lead will be assessed for reduction of A?42 levels in the APPJ2O mouse model of AD. The development of selective chemical activators of autophagy will serve as highly valuable molecular probes that we anticipate will reveal new insights into the regulation of this key cellular process and its role in the pathophysiology of AD.